Summary Biofilms, multicellular bacterial communities, are associated with up to 65% of human infections. Amyloid proteins and DNA decorate the extracellular matrix (ECM) of the biofilms produced by both Gram- positive and Gram-negative bacteria. This ECM encapsulates the bacteria and is primarily responsible for the recalcitrance of bacteria to antimicrobial substances and immune host defenses. Toll-like receptors (TLRs) and NOD-like receptors (NLRs), orchestrate appropriate immune responses to bacteria and viruses through the recognition of conserved molecular patterns. Our group has shown that curli amyloid fibers, found in the Gram-negative enteric biofim ECM, complexed with DNA serve as a molecular pattern for the immune system. Curli complexed with DNA powerfully stimulates innate immune cells through a two-step mechanism: First, curli activates the TLR2/1 heterocomplex, and then internalization of this TLR2/1 complex brings curli/DNA into the endosome to activate the DNA receptor TLR9. Activation of TLR2 and TLR9 results in the generation of a strong proinflammatory response and the generation of type I interferons. Intriguingly, these interactions also result in an autoimmune response. In fact, Gram-negative bacteria expressing the amyloid curli or systemic exposure to purified curli/DNA complexes trigger the production of anti-double-stranded DNA/anti-chromatin autoantibodies, suggesting that curli/DNA complexes trigger autoimmunity during infections. Intriguingly, our preliminary data shows that the PSM amyloids from Gram-positive S. aureus that bind DNA in the ECM also trigger such responses. We hypothesize that biofilms trigger an autoimmune response due to activation of TLR2 and TLR9 by bacterial amyloid/DNA complexes. We will study biofilms of two common human pathogens, E. coli and Staphylococcus aureus as prototypical Gram-negative and Gram-positive bacteria, respectively. In Aim1, we will determine how the innate immune system recognizes bacterial amyloid/DNA complexes from Gram- positive vs Gram-negative bacteria In Aim 2, we will establish biofilm infections and study the immune responses generated against the biofilm and the subsequent development of autoimmunity.